High affinity adaptor molecules for redirecting antibody specifity

ABSTRACT

Disclosed are methods for identifying high affinity adaptor molecules that bind to both a circulating antibody and a target molecule and redirect the specificity of the circulating antibody to the target molecule. Exemplary high affinity adaptor molecules are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/248,778, filed Oct. 5, 2009, and U.S. Provisional ApplicationSer. No. 61/257,351, filed Nov. 2, 2009. The entire contents of theaforementioned applications are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The concept of redirecting the immune system to attack new targets haslong interested scientists as an appealing strategy for targetedimmunotherapy. By redirecting naturally circulating human antibodies toattack desired targets in disease areas such as cancer, autoimmunedisease and infectious disease, one can avoid the need for a specialimmunization. While this strategy has shown early signs of success, moststudies have not progressed beyond in vitro demonstrations. For example,this strategy would be particularly valuable if it made use of antibodyalready present in the general population and could be made amenable tooral administration. Thus, there is a need in the art for improvedmethods of redirecting antibody specificity.

SUMMARY OF THE INVENTION

The present invention provides isolated adaptor molecules, particularlybispecific adaptor peptides, which bind to both an antibody and adesired target molecule with high binding affinity and selectivity. Dueto their high binding affinity and selectivity, the adaptor molecules ofthe invention are capable of efficiently redirecting circulatingantibodies to a target molecule not normally bound by the antibodymolecule. Moreover, the adaptor molecules disclosed herein provide oneor more of the following advantages over traditional antibody-mediatedtherapeutics: 1) enable the simultaneous recruitment of multipleantibody-class effector functions; 2) can be developed rapidly using themethods of the invention; 3) have a low cost of goods; and 4) do notcause an IgE-mediated hypersensitivity reaction when administered to asubject.

The adaptor molecules generally comprise one or more targeting moieticslinked to one or more ligand moietics. In certain embodiments, theligand moiety comprises one or more Gal antigen (e.g., Gal-α-1-3-Gal) ormimetic thereof. In one embodiment, the targeting moiety comprises apeptide, e.g., a VEGF or TNFα-binding peptide. Exemplary VEGF-bindingpeptides have one or more of the amino acid sequences set forth in SEQID NO. 1, 2, 3, and/or 4. In another preferred embodiment, the peptide(e.g., the peptide sequence set forth in SEQ ID NO. 1, 2, 3, and/or 4)is linked to a ligand moiety comprising one or more Gal antigens (e.g.,Gal-α-1-3-Gal disachharide).

In other embodiments, the targeting moiety comprises one or moreantibody, or antigen binding fragment thereof. Suitable antibodiesinclude, without limitation, Abciximab, Adalimumab, Alemtuzumab,Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol, Daclizumab,Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab,Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab,Ranibizumab, Rituximab, Tositumomab, Trastuzumab, and/or Golimumab, orantigen binding fragments thereof. In a preferred embodiment, theantibody (e.g., one or more of the antibodies disclosed supra) orantigen binding fragments thereof, is linked to a ligand moietycomprising one or more Gal antigens (e.g., Gal-α-1-3-Gal). In anotherpreferred embodiment, the antibody (e.g., one or more of the antibodiesdisclosed supra) or antigen binding fragments thereof, is linked to aligand moiety comprising one Gal-α-1-3-Gal disaccharide. In anotherpreferred embodiment, a ligand moiety comprising one or more Galantigens (e.g., Gal-α-1-3-Gal) is linked to one or more variable regionsof the antibody.

In other embodiments, the targeting moiety comprises an antibody-likemolecule. Suitable antibody-like molecules include, without limitation,Adnectins, Affibodies, DARPins, Anticalins, Avimers, and Versabodies, orantigen binding fragment thereof. In a preferred embodiment, theantibody-like molecule is linked to a ligand moiety comprising one ormore Gal antigens (e.g., Gal-α-1-3-Gal).

In other embodiments, the targeting moiety of the invention comprises anextracellular portion of a cell surface receptor, or fragment thereof.Suitable cell surface receptors include, without limitation, a TNFfamily receptor (e.g., a TNFα receptor, e.g., a human TNFα receptor) andgrowth factor receptors of the tyrosine kinase family, (e.g, p185HER2).In a preferred embodiment, the extracellular portion of a cell surfacereceptor molecule is linked to a ligand moiety comprising one or moreGal antigens (e.g., Gal-α-1-3-Gal). In another preferred embodiment, theextracellular portion of a cell surface receptor molecule is linked to aligand moiety comprising one Gal-α-1-3-Gal disaccharide.

In other embodiments, the targeting moiety comprises a ligand for a cellsurface receptor. In a preferred embodiment, the ligand is linked to aligand moiety comprising one or more Gal antigens (e.g., Gal-α-1-3-Gal).In another preferred embodiment, the ligand is linked to a ligand moietycomprising one Gal-α-1-3-Gal disaccharide.

In another aspect, the invention also provides methods for identifyingisolated adaptor molecules, such methods comprising: providing arandomized library encoding a population of candidate targetingmoietics; selecting a targeting moiety from the display library whichbinds with high affinity and/or selectivity to a target molecule;linking the targeting moiety to a ligand moiety via a linking moiety toform a candidate adaptor molecule; and evaluating the ability of thecandidate adaptor molecule to redirect the specificity of thecirculating antibody to the target molecule. Suitable screening methodsfor use in the methods of the invention include an mRNA display,ribosome display, yeast display, phage display or screening of syntheticpeptide library. In one preferred embodiment, an mRNA display library isused to select a targeting peptide. In another preferred embodiment, aphage display library is used to select a targeting peptide.

A further aspect of the invention provides a method of treating adisease (e.g., a cancer, an infectious disease, or an autoimmunedisease) in a subject, comprising administering to the subject aneffective amount of an isolated adaptor molecule of the invention,thereby treating the disease. In specific embodiments the disease is atleast one of macular degeneration, diabetic retinopathy, psoriasis,diabetes, cardiovascular ischemia, rheumatoid arthritis, andosteoarthritis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic overview of an exemplary adaptor molecule of theinvention. The adaptor molecule is a bispecific peptide comprising ahigh-affinity peptide targeting moiety directed against a target ofinterest and a ligand moiety comprising the glycopeptide epitope of ananti-gal antibody. By binding to both the target molecule and anaturally-existing anti-gal antibody, the adaptor molecule is capable ofredirecting the effector functions of the antibody to act on the targetmolecule.

FIG. 2 provides an overview of the various method steps conducted duringmRNA display.

FIG. 3 depicts exemplary peptide libraries that may be used in mRNAdisplay for selection of high affinity adaptor peptides.

FIG. 4 depicts four exemplary high affinity VEGF targeting peptides ofthe invention.

FIG. 5 depicts exemplary methods and linkers for coupling a targetmoiety (e.g., a VEGF targeting moiety of the invention) to a ligandmoiety.

FIG. 6 depicts HPLC and Mass spectrometry analyses of a targetmoiety/ligand moiety coupling reaction.

FIG. 7 depicts HPLC and Mass spectrometry analyses of an optimizedtarget moiety/ligand moiety coupling reaction.

FIG. 8 depicts the results of in vitro assays demonstrating the efficacyof VEGF-binding adaptors of the invention at redirecting naturallyoccurring anti-gal antibodies to bind VEGF.

DETAILED DESCRIPTION

This specification describes, inter alia, the identification andproduction of novel, adaptor molecules that bind to both antibodies andtarget molecules with high binding affinity and selectivity. As usedherein, the term “adaptor” refers to the ability of the peptide tofacilitate a functional interaction between an antibody and a targetmolecule to which the antibody does not normally bind. For example, theadaptor molecules of the invention are capable of binding to both anantibody and a target molecule such that the antibody and target arebrought into close proximity with each other. In certain embodiments,the antibody is capable of facilitating an antibody response against thetarget molecule. Exemplary antibody responses may include neutralizationor opsonization of a target molecule (e.g.., a soluble factor such as acytokine or growth factor). Alternatively, where the target molecule ispresent on the surface of virus, the antibody may facilitateneutralization or opsonization of the virus. In other embodiments, thetarget molecule may present on the surface of a cell (e.g., an infectedcell or tumor cell) and recruitment of the antibody to the cell by theadaptor peptide facilitates the induction of an antibody-mediatedeffector response (e.g., induction of a complement carcase orantibody-dependent cellular cytotoxicity (ADCC)). The adaptor moleculesdisclosed herein are particularly advantageous in that they do notappreciably activate basophils, and therefore do not cause an IgEhypersensitivity reaction when administered to a subject.

Adaptor molecules of the invention comprise at least three moieties: (a)a targeting moiety, (b) a ligand moiety and (c) a linker moiety. Thetargeting moiety (a) is a moiety which binds with high affinity and/orselectivity to a target molecule. The ligand moiety (b) is a moiety towhich a circulating antibody binds with high affinity and/orselectivity. The targeting moiety and ligand moiety are operably linkedvia the intervening linker moiety (c). Said linker moiety may be acovalent bond, chemical linker, or peptide amino acid sequence, or anyother moiety capable of linking the targeting and ligand moieties of theadaptor peptide.

(a) Targeting Moiety

A targeting moiety of the invention has been selected for its ability tobind with high affinity and/or selectivity to a target molecule. Inparticular embodiments, the targeting moiety specifically binds to thetarget molecule with a dissociation constant (ICD) of 100 nanomolar orless (e.g., 10 nM or less, 1 nM or less, 100 pM or less, 10 pM or less,or 1 pM or less). In other embodiments, the targeting moiety exhibitshigh selectivity. By “specifically binds” is meant that the moietyrecognizes and interacts with a target molecule but that does notsubstantially recognize and interact with other molecules in a sample,e.g., a biological sample. In particular embodiments, the bindingaffinity of the targeting moiety for the target molecule is at least1000 fold higher than its binding affinity for a non-target molecule(e.g., 10³ fold, 10⁴ fold, 10⁵ fold, 10⁶ fold, or 10⁷ fold higher).

Targeting moieties can be selected for the ability to bind with highselectivity and/or affinity to virtually any target molecule. In certainembodiments, the targeting moiety binds to a pathogen-associated targetmolecule, including, but not limited to, surface proteins or antigensfrom a virus (e.g., HAV, HBV, or HCV, HIV, influenza virus), bacteria,yeast, parasites, or fungus. In other embodiments, the target moleculeis a cell surface protein, including but not limited to, a cell surfaceantigen or receptor from an infected host cell or a tumor cell.Exemplary tumor-associated antigens include the growth factor receptorof the tyrosine kinase family, p185HER2. In other embodiments, thetarget molecule is a hormone or growth factor. Exemplary hormones orgrowth factors include tumor necrosis factor alpha (TNFα) or Vascularendothelial growth factor (VEGF). In other embodiments, the targetmolecule is an antibody (e.g., an auto-antibody).

In one preferred embodiment, the targeting moiety of the inventioncomprises a peptide moiety. The length of the peptide targeting moietyis desirably between at least 3-200 amino acids, preferably between atleast 3-100 amino acids, more preferably between 3-50 amino acids, andstill more preferably between 3-30 amino acids (e.g., 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 amino acids). Exemplary peptides for use as targetingmoieties are described in WO/2010/014830, which is herein incorporatedby reference in its entirety. In a preferred embodiment, the peptide isa VEGF-binding peptide comprising or consisting of any one or more ofthe following amino acid sequences, or VEGF-binding portions thereof:

SEQ ID NO. 1 H-Gly-Val-Gln-Glu-Asp-Val-Ser-Ser-Thr-Leu-Gly-Ser-Trp-Val-Leu-Leu-Pro-Phe-His-Arg-Gly-Thr-Arg-Leu- Ser-Val-Trp-Val-ThrSEQ ID NO. 2 H-Gly-Gly-Phe-Glu-Gly-Leu-Ser-Gln-Ala-Arg-Lys-Asp-Gln-Leu-Trp-Leu-Phe-Leu-Met-Gln-His-Ile-Arg-Ser- Tyr-Arg-Thr-Ile-ThrSEQ ID NO. 3 H-Gly-Val-Gly-Gly-Ser-Arg-Leu-Glu-Ala-Tyr-Lys-Lys-Asp-His-Arg-Val-Phe-Gln-Met-Ala-Trp-Leu-Gln-Tyr- Tyr-Trp-Ser-Thr-Thr;and/or SEQ ID NO. 4 H-Gly-Ser-Gly-Ser-Gly-Asn-Ala-Leu-His-Trp-Val-Cys-Ala-Ser-Asn-Ile-Cys-Trp-Arg-Thr-Pro-Trp-Ala-Gly-Gln-Leu-Trp-Gly-Leu-Val-Arg-Leu-Thr.

In other embodiments, the targeting moiety of the invention comprises anantibody, or binding fragment thereof (e.g., a CDR (e.g., CDRH3), avariable domain (VH or VL), or a Fab fragment). Any antibody, orfragment thereof, from any animal species, is contemplated for use inthe methods and compositions described herein. Suitable antibodies andantibody fragments include, without limitation, single chain antibodies(see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.(1988) Proc. Natl. Acad. Sci. U.S.A 85:5879-5883, each of which isherein incorporated by reference in its entirety), domain antibodies(see, e.g., U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197;6,696,245, each of which is herein incorporated by reference in itsentirety), Nanobodies (see, e.g., U.S. Pat. No. 6,765,087, which isherein incorporated by reference in its entirety), and UniBodies (see,e.g., W02007/059782, which is herein incorporated by reference in itsentirety In certain embodiments, the antibody is Abciximab, Adalimumab,Alemtuzumab , Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol,Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan,Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab,Panitumumab, Ranibizumab, Rituximab, Tositumomab, Trastuzumab, and/orGolimumab, or antigen binding fragments thereof.

In other embodiments, the targeting moiety of the invention comprises anantibody-like molecule. Suitable antibody-like molecules include,without limitation, Adnectins (see, e.g., WO 2009/083804, which isherein incorporated by reference in its entirety), Affibodies (see,e.g., U.S. Pat. No. 5,831,012, which is herein incorporated by referencein its entirety), DARPins (see, e.g., U.S. Patent ApplicationPublication No. 2004/0132028, which is herein incorporated by referencein its entirety), Anticalins (see, e.g., U.S. Pat. No. 7,250,297, whichis herein incorporated by reference in its entirety), Avimers (see,e.g., U.S. Patent Application Publication Nos. 200610286603, which isherein incorporated by reference in its entirety), and Versabodies (see,e.g., U.S. Patent Application Publication No. 2007/0191272, which ishereby incorporated by reference in its entirety).

In other embodiments, the targeting moiety of the invention comprises aligand for a cell surface receptor, wherein the ligand is capable ofrecruiting the adaptor molecule to cells that express said cell surfacereceptor.

In other embodiments, the targeting moiety of the invention comprisesthe extracellular portion of a cell surface receptor, or fragmentthereof, wherein the cell surface receptor, or fragment thereof, iscapable of recruiting the adaptor molecule to the cognate ligand of saidcell surface receptor. Suitable cell surface receptors include, withoutlimitation, TNF family receptors (e.g., a TNFα receptor, e.g., a humanTNFα receptor) and growth factor receptors of the tyrosine kinasefamily, (e.g, p185HER2).

In certain exemplary embodiments, the targeting moiety of the inventioncomprises an Fc fusion protein or immunoadhesin (e.g., a TNF receptor-Fcfusion such as Etaneracept).

(b) Ligand Moiety

Ligand moieties of the invention comprise antigenic domains which arebound by Immunoadhesions (Fc fusions proteins) present in a subject. Insome embodiments, the ligand moiety is bound by a circulating antibody.Circulating antibodies may be present in the subject due to naturallyacquired immunity. Alternatively, the circulating antibodies are presentas a result of prior vaccination of the subject. For example, thecirculating antibodies may be present as a result of childhoodvaccination against small pox, measles, mumps, rubella, herpes,hepatitis and polio. Accordingly, a ligand moiety may comprise one ormore epitopes that is recognized by these circulating antibodies.

In some embodiments, however, a ligand moiety interacts with an antibodythat has been administered to the subject. For example, an antibody thatinteracts with the ligand moiety of an adaptor molecule of the inventioncan be co-administered with the adaptor molecule. Further, the antibodythat interacts with ligand moiety may not normally exist in a subjectbut the subject has acquired the antibody by introduction of a biologicmaterial or antigen (e.g., serum, blood, or tissue) so as to generate ahigh titer of antibodies in the subject. For example, subjects thatundergo blood transfusion acquire numerous antibodies, some of which caninteract with a ligand moiety of the adaptor peptide.

A ligand moiety can comprise any compound capable of binding to anantibody, including, without limitation, a peptide, carbohydrate, lipid,antibody, or antibody-like molecule. In some embodiments, a ligandmoiety (e.g., a peptide, antibody or antibody-like molecule) cancomprise one or more non-natural amino acids. Preferably, the ligandmoiety comprises an epitope that binds to a “high-titer antibody.” Theterm “high-titer antibody” as used herein, refers to an antibody thathas high affinity for an antigen (e.g., an epitope on an antigenicdomain). For example, in a solid-phase enzyme linked immunosorbent assay(ELISA), a high titer antibody corresponds to an antibody present in aserum sample that remains positive in the assay after a dilution of theserum to approximately the range of 1:100-1:1000 in an appropriatedilution buffer. Other dilution ranges include 1:200-1:1000,1:200-1:900, 1:300-1:900, 1:300-1:800, 1:400-1:800, 1:400-1:700,1:400-1:600, and the like. In certain embodiments, the ratio between theserum and dilution buffer is approximately: 1:100, 1:150, 1:200, 1:250,1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750,1:800, 1:850, 1:900, 1:950, 1:1000.

In certain embodiments, the ligand moieties are antigenic peptidesobtained from a known target molecule (e.g., a surface protein from apathogen, tumor cell, or infected host cell) of the antibody. The lengthof the peptide ligand moiety is desirably between at least 3-200 aminoacids, preferably between at least 3-100 amino acids, more preferablybetween 3-50 amino acids, and still more preferably between 10-25 aminoacids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids). In someembodiments, the peptides are comprised of natural amino acids. In otherembodiments, the peptides include one or more non-natural amino acids(e.g., D-amino acids).

In certain embodiments, the ligand moiety is a glycosylated ligandmoiety. Glycosylated ligand moietics can comprise or consist of anantigenic saccharide or glycan moiety recognized by an antibody in asubject. In some embodiments, the glycosylated ligand moiety is linkeddirectly or indirectly (i.e., via a linker moiety) to the targetingmoiety of the adaptor molecule. Such ligand moieties lack an antigenicpeptide moiety. In other embodiments, the glycosylated ligand moiety isa glycopeptide comprising additional antigenic peptide elements (e.g.,an antigenic domain comprising a peptide or epitope of a pathogen).

Exemplary glycosylated ligand moieties are derived from blood groupantigens. These antigens are generally surface markers located on theoutside of red blood cell membranes. Most of these surface markers areproteins, however, some are carbohydrates attached to lipids orproteins. Structurally, the blood group determinants that can be usedwith the embodiments described herein fall into two basic categoriesknown as type I and type II. Type I comprises a backbone comprised of agalactose 1-3 β linked to N-acetyl glucosamine while type II comprises,instead, a 1-4 β linkage between the same building blocks. The positionand extent of fucosylation of these backbone structures gives rise tothe Lewis-type and H-type specificities. For example, the presence of ana -monofucosyl branch, solely at the C2-hydroxyl in the galactose moietyin the backbone, constitutes the H-type specifity (Types I and II),while further permutation by substitution of a-linked galactose ora-linked N-acetylgalactosamine provides the molecular basis of thefamiliar serological blood group classifications A, B, and O. By firstdetermining a patient's particular set of blood group antigens, one canselect a ligand moiety comprising one or more blood group antigens thatare outside of the repertoire of the patient so as to generate a potentresponse to an adaptor molecule comprising this ligand moiety andthereby redirecting the antibodies present in the patient to targetmolecule bound by the targeting moiety of said adaptor molecule.Exemplary blood group antigens are set forth in detail in Table 2 ofU.S. Pat. No. 7,318,926, which is hereby incorporated by reference inits entirety.

In certain preferred embodiments, the glycosylated ligand moietycomprises one or more gal-α-1-3 gal disaccharide sugar units of the galantigen. The gal antigen is produced in large amounts on the cells ofpigs, mice and New World monkeys by the glycosylation enzymegalactosyltransferase (α(1,3)GT). Since humans and Old World primateslack the gal antigen, they are not immunotolerant to it and produceanti-gal antigen antibodies (anti-Gal) throughout life in response toantigenic stimulation by gastrointestinal bacteria. It has beenestimated that anti-gal antibodies represent more than 2% of circulatingIgG and 1-8% of circulating IgM in humans. The binding of anti-Gal togal antigens expressed on glycolipids and glycoproteins on the surfaceof endothelial cells in donor organs leads to activation of thecomplement cascade and hyperacute rejection, and also plays an importantrole in occurrence of complement-independent delayed xenograftrejection. Accordingly, the gal antigen has the ability to generate apotent immune response.

In certain preferred embodiments, the glycosylated ligand moiety to bejoined or incorporated into the adaptor molecule consists essentially ofone or more Gal-α(1-3)-Gal disaccharide sugar units and lacks any of theremaining portions of the Gal antigen (e.g., GlcNac or Glc). Forexample, one or more Gal-α(1-3)-Gal disaccharides may be linked to atargeting moiety of an adaptor molecule via a free hydroxyl group at thereducing end of a disaccharide unit (e.g., the hydroxyl group at the C1carbon of that is not involved in a glycosidic bond). In certainembodiments the Gal disaccharide is linked to the targeting moiety via aspacer moiety (e.g., C1-C6 alkyl spacer) at the free hydroxyl group.Without being limited to any particular theory, it is thought that theGal-α(1-3)-Gal disaccharide portion of the Gal antigen bindspreferentially to anti-Gal antibodies but exhibits limited binding tolectins (e.g., Galectin-3) or other molecules that bind to otherportions of the Gal antigen (e.g., the Gal-β(1-4)-GlcNAc moiety). Inother embodiments, the glycosylated ligand moiety to be joined orincorporated into adaptor molecule comprises additional sugar residuesof the Gal antigen. For example, one or more trisaccharide(Gal-α(1-3)-Gal-β(1-4)-GlcNAc or Gal-α(1-3)-Gal-β(1-4)-Glc),tetrasccharide (Gal-α(1-3)-Gal-β(1-4)-GlcNAc-β(1-3)-Gal) orpentasaccharide (Gal-α(1-3)-Gal-β(1-4)-GlcNAc-β(1-3)-Gal-β(1-4)-Glc)units of the gal antigen may incorporated.

In certain embodiments the gal antigen to be joined or incorporated intoan adaptor molecule is selected from gal-α-(1,3) gal series ofneoglycoproteins and can include: Gal α 1-3Gal-BSA (3-atom spacer), Galα 1-3Gal-BSA (14-atom spacer), Gal α 1-3Gal-HSA (3-atom spacer), Gal α1-3Gal-HSA (14-atom spacer), Gal α 1-3Galβ1-4GlcNAc-BSA (3-atom spacer),Galα1-3Galβ1-4GlcNAc-BSA (14-atom spacer), Gal α 1-3Galβ1-4GlcNAc-HSA(3-atom spacer), Gal α 1-3Galβ1-4GlcNAc-HSA (14-atom spacer), Galα1-3Gal-PentasaccharideBSA (3-atom spacer), and the like. In otherembodiments the gal antigen can be selected from gal α (1,3) galanalogue neoglycoproteins, including Gal α 1-3Galβ1-4Glc-BSA (3-atomspacer), Gal α 1-3Galβ1-4Glc-HSA (3-atom spacer),Gala1-3Galβ1-3GlcNAc-BSA (3-atom spacer), Gal α 1-3Galβ1-3GlcNAc-HSA(3-atom spacer), Gal α 1-3Galβ1-4(3-deoxyGlcNAc)-HSA (3-atom spacer),Gal α 1-3Galβ1-4(6-deoxyGlcNAc)-HSA, and the like.

In yet other embodiments, a peptidomimetic of a Gal antigen can beincorporated into an adaptor molecule of the invention. Exemplarypeptidomimetics include the αGal-linked glycopeptides Gal-α-YWRY,Gal-α-TWRY and Gal-α-RWRY. Other peptidomimetics can be identified byscreening a randomized library of αGal glycopeptides for anti-Galantibody binding activity using the methods of Xian et al. (see J. Comb.Chem., 6:126-134 (2004), which is incorporated by reference herein).

A Gal antigen, or peptidomimetic thereof, can be linked to a targetingmoiety (covalently and/or non-covalently) using any art-recognizedmeans. Art-recognized non-covalent linkages include biotin-avidin orbiotin-streptavidin linkages and other high affinity binding partners(e.g., leucine zippers and the like). Suitable means for covalentattachment include, without limitation, those set forth in FIG. 4 and USPatent Application number 20100183635, which is hereby incorporated byreference in its entirety. For example the gal antigen (orpeptidomimeitc) may be linked directly to the polypeptide backbone of apolypeptide targeting moiety via a synthetic chemical linker. Exemplarysynthetic chemical linkers include bifunctional linker moieties, e.g.,linkers with maleimide functionality. For example, the bifunctionallinker moiety may link the targeting moiety and the ligand moiety via anamino group in the targeting moiety and a thiol moiety in the ligandmoiety, or vice versa. In one exemplary emobodiment, a maleimide linker(e.g., Sulfo-SMCC) is used to link a cysteine residue of a polypeptidetargeting moiety to the amino group of an amino modified Gal antigen(e.g., B_(di)—(CH₂)₃—NH₂ of FIG. 5). Additionally or alternatively, thegal antigen may be linked to N-linked oligosaccharide of a glycoproteintargeting moiety. In some embodiments, one or more Gal antigens (e.g.,gal-α-(1,3) gal), or peptidomimetics thereof, are linked to a singlesite on a target moiety. In other embodiments, one or more Galantigens.(e.g., gal-α-(1,3) gal), or peptidomimetic thereof, are linkedto multiple sites on a target moiety. In certain embodiments, the linkermoiety is chemically-modified to reduce enzymatic or chemicaldegradation.

Adaptor molecules comprising Gal antigen as disclosed herein areparticularly advantageous in that they do not appreciably activatebasophils, and therefore do not cause an IgE-mediated hypersensistivityreaction when administered to a subject. Notwithstanding, in someembodiments the adaptor molecules of the invention will be assayed fortheir ability to activate basophils. Suitable assays for measuringbasophil activation are known in the art, (see, e.g., J. Allergy ClinImmunol (2002) 110102 -9, which is hereby incorporated by reference inits entirety).

In certain embodiments, the ligands moiety comprises polymeric bindingmolecules wherein the monomers are not amino acids.

In certain exemplary embodiments, the high affinity adaptor molecule isselected from the group consisting of:

(a) H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-Val-D-Trp-D-Val-D-Thr-PEG₂-Cys-X-Y;(b) H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-Arg-D-Thr-D-Ile-D-Thr-PEG₂-Cys-X-Y;(c) H-Gly-D-Val-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-Trp-D-Ser-D-Thr-D-Thr-PEG₂-Cys-X-Y; and(d) H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr- PEG2-Cys-X-Y;

wherein, X is bifunctional chemical linker with maleimide functionality;and Y is an amino modified Gal-1-3-Gal disaccharide.

(c) Modified Adaptor Molecules

One or more moieties of the adaptor molecules of the invention may bemodified. In certain embodiments, a peptide moiety of the adaptormolecule is modified. For example, peptide moieties of an adaptormolecule can be modified to include non-natural amino acids such asthose described in U. S. Pat. No. 6,559,126, incorporated herein byreference. For example, the peptides of the invention may be composed ofone or more, or most preferably all, amino acids which are D-typeoptical isomers. These D-peptides have several advantages with respectto antibodies and other protein therapeutics. The smaller size andgreater stability of the D-peptides makes them simpler to formulate forpulmonary, topical and oral delivery. D-peptides are also known to bepoor immunogens (Dintzis et al. (1993) PROTEINS: Structure, Function,and Genetics 16, 306-308). Furthermore, their resistance to enzymaticdegradation, and their ability to be combined with polymers, results inenhanced pharmacokinetics compared to other peptide drugs. Also,D-peptides have reduced manufacturing costs that could be passed on tothe consumer.

The peptide component of an adaptor molecule can also be modified by anyvariety of standard chemical methods (e.g., an amino acid can bemodified with a protecting group; the carboxy-terminal amino acid can bemade into a terminal amide group; the amino-terminal residue can bemodified with groups to, e.g., enhance lipophilicity; or the polypeptidecan be chemically glycosylated or otherwise modified to increasestability or in vivo half-life). Adaptor molecules of the invention maybe designed to include chemical modifications or particular amino acidsequences which promote solubility. For example, in some embodimentspeptide moieties may be synthesized to include the amino acids DDD orKKK in the N-terminal or C-terminal regions. Additionally oralternatively, peptides and other targeting moieties may be synthesizedto include a PEGylation moiety at, for example, the N-terminal and/or —Cterminal regions. Exemplary PEGylation moieties include PEG₂-NH2 andPEG₂-Cys-NH2 moieties.

The present invention also encompasses “conservative sequencemodifications” or “conservative amino acid modifications” of thesequences described herein, i.e., amino acid sequence modificationswhich do not significantly affect or alter the binding characteristicsof the peptide encoded by the nucleotide sequence or containing theamino acid sequence. Such conservative sequence modifications includenucleotide and amino acid substitutions, additions and deletions.Modifications can be introduced into sequences by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. In some embodiments, the modifications are chosen byrational design, and the designed peptides are generated by chemicalsynthesis as described herein. “Conservative amino acid modifications”includes conservative amino acid substitutions which are substitutionsin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain (e.g., similar size, shape, electric charge,chemical properties including the ability to form covalent or hydrogenbonds, or the like). Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

A peptide or mimetic thereof of the invention may be modified by one ormore substitutions, particularly in portions of the protein that are notexpected to interact with a target protein. It is expected that as manyas 5%, 10%, 20%, 30%, 40%, 50%, or even 50% or more of the amino acidsin peptide may be altered by a conservative substitution withoutsubstantially altering the affinity of the protein for target. It may bethat such changes will alter the immunogenicity of the polypeptide invivo, and where the immunogenicity is decreased, such changes will bedesirable. Further non-limiting examples of homologous substitutionsthat can be made in the structures of the peptidic molecules of theinvention include substitution of D-phenylalanine with D-tyrosine,D-pyridylalanine or D-homophenylalanine,.substitution of D-leucine withD-valine or other natural or non-natural amino acid having an aliphaticside chain and/or substitution of D-valine with D-leucine or othernatural or non-natural amino acid having an aliphatic side chain. Insome embodiments, conservative amino acid substitutions alone, i.e.,without amino acid deletions or additions are the preferred type ofamino acid modification. One of skill in the art will appreciate thatsuch modifications or substitutions may be made at the DNA level, thusencoding the altered or substituted peptide, or they may be made at theprotein level, e.g., by direct chemical synthesis.

In some embodiments a peptide or peptide moiety of an adaptor moleculemay be made cyclic. Such “cyclic peptides” have intramolecular linkswhich connect two amino acids. Cyclic peptides are often resistant toproteolytic degradation and are thus good candidates for oraladministration. The intramolecular linkage may encompass intermediatelinkage groups or may involve direct covalent bonding between amino acidresidues. In some embodiments, the N-terminal and C-terminal amino acidsare linked. In other embodiments, one or more internal amino acidsparticipate in the cyclization. Other methods known in the art may beemployed to cyclize peptides of the invention. For example, cyclicpeptides may be formed via side-chain Azide-Alkyne 1,3-dipolarcycloaddition (Cantel et al. J. Org. Chem., 73 (15), 5663-5674, 2008,incorporated herein by reference). Cyclization of peptides may also beachieved, e.g., by the methods disclosed in U.S. Pat. Nos. 5,596,078;4,033,940; 4,216,141; 4,271,068; 5,726,287; 5,922,680; 5,990,273;6,242,565; and Scott et al. PNAS. 1999. vol. 96 no. 24 P. 13638-13643,which are all incorporated herein by reference. In some embodiments theintramolecular link is a disulfide bond mimic or disulfide bond mimeticwhich preserves the structure that would be otherwise be created by adisulfide bond.

In some particularly preferred embodiments, the cyclization of peptidesoccurs via intramolecular disulfide bonds. In some preferredembodiments, the formation of an intramolecular disulfide bond increasesthe affinity of the peptide. Accordingly, the methodology used to selectand/or affinity mature the peptides or mimetics thereof of the inventionmay be performed under conditions which allow disulfide bond formationprior to and during selection (e.g., oxidizing conditions). In someparticularly preferred embodiments the disulfide bonds may form betweencysteine residues which naturally exist in the library or peptide, orwhich are introduced by the mutation process during one or more roundsof selection. In other embodiments the peptides may be designed tocontain cysteine residues at particular positions such that it is knownwhich residues participate in the disulfide bond. Intramoleculardisulfide bonding between cysteine residues may be induced by methodsknown in the art (e.g., U.S. Pat. Nos. 4,572,798; 6,083,715; 6,027,888,and WIPO Publication WO/2002/103024 which are incorporated herein byreference).

In some embodiments, the formation of a disulfide bond (or the formationof a cyclized or intramolecularly linked structure in general) imparts aparticular structure onto the peptide which is important for targetbinding. Accordingly, the disulfide bonds and/or cyclization preferablyform prior to peptide selection such that the potentially favorablestructure created by bond formation may be selected for. In someembodiments the peptides or mimetics thereof of the invention may havemore than one, two, three, or more disulfide bonds. Further methodsknown in the art to generate, and select peptides with intramoleculardi-sulfide bonds, intramolecular di-sulfide bond substitutes, and otherintramolecular links may be employed. For example, the methods describedin WO03040168, incorporated herein by reference, describe methods togenerate and select peptide apatamers, conotides, and other cyclicpeptides which, in some embodiments, may be employed with the methods ofthe present invention.

In related embodiments a peptide conformation or structure which isbeneficial to binding (e.g., it increases binding affinity) may bepreserved or mimicked by chemical crosslinking or other methods ofpeptide stabilization. For example, a beneficial peptide conformation orstructure which is formed by disulfide bonds may be stabilized bychemical treatment or reaction, thus allowing the preservation of thestructure without a disulfide bond. Indeed, peptide stabilizationtechniques may be employed to stabilize peptides of the inventionwhether or not a disulfide bond was originally present. For example, thetechniques described in Jackson, et al. J. Am. Chem. Soc. 1991, 113,9391-9392; Phelan, et al. J. Am. Chem. Soc. 1997, 119, 455-460; Bracken,et al. J. Am. Chem. Soc. 1994, 116, 6431-6432, which are incorporatedherein by reference, may be used to stabilize peptides or peptidemoieties of the invention.

Other methods to stabilize peptides and peptide structures may be used,e.g., olefinic cross-linking of helices through O-allyl serine residues(Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37,3281-3284, incorporated herein by reference), all-hydrocarboncross-linking (Schafmeister and Verdine J. Am. Chem. Soc. 2000, 122(24), 5891-5892, incorporated herein by reference) and the methodsdisclosed in U.S. Pat. No. 7,183,059 (incorporated herein by reference).The methods disclosed in Blackwell et al. and Schafmeister et al. may bedescribed as producing “stapled” peptides, i.e., peptides which arecovalently locked into a particular conformational state or secondarystructure, or peptides which have a particular intramolecular covalentlinkage which predisposes them to form a particular conformation orstructure. If a peptide thus treated is predisposed to, e.g., form analpha-helix which is important for target binding, then the energeticthreshold for binding will be lowered. Such “stapled” peptides have beenshown to be resistant to proteases and may also be designed to cross thecellular membrane more effectively (also see Walensky et al. Science2004: Vol. 305. no. 5689, pp. 1466-1470; Bernal et al. J Am Chem Soc.2007, 129(9):2456-7 which are incorporated herein by reference).Accordingly, peptides or peptide moieties of the invention may be thusstapled or otherwise modified to lock them into a specificconformational shape or they may be modified to be predisposed toparticular conformation or secondary structure which is beneficial forbinding. It is contemplated that such peptide modifications may occurprior to peptide selection such that the benefit of any conformationalconstraints may also be selected for. Alternatively, in someembodiments, the modifications may be made after selection to preserve aconformation known to be beneficial to binding or to further enhance apeptide candidate.

In other embodiments, the ligand moiety of an adaptor molecule can bemodified. Said modifications can be made, for example, to minimizecompetitive binding by interfering molecules or reduce enzymatic orchemical degradation of the ligand moiety (e.g., under physiologicalconditions). As used herein, the term “interfering molecule” refers to abinding molecule (e.g., a circulating or cell surface receptor) thatcompetes with circulating antibodies for binding to an adaptor moleculeand prevents it from exerting its intended therapeutic effect (e.g., byrapidly clearing the adaptor molecule from circulation). For example, aglycosylated ligand moiety may comprise a gal antigen or mimetic thathas been chemically-modified to enhance preferential binding by anti-Galantibodies while minimizing undesirable binding by a lectin (e.g.,Galectin-3) or other interfering molecule. Additionally oralternatively, a glycosylated ligand moiety may comprise a gal antigenor mimetic that has been chemically-modified, for example, to reduceenzymatic or chemical degradation or facilitate covalent linkage.Exemplary modifications include the addition of biologically inertprotecting groups to reactive hydroxyl groups on the sugar residues of agal antigen, e.g., via dehydrative coupling, reductive amination, orenzymatic oxidation, e.g., with galactose oxidase. Protecting groups maybe added to the C-6′ OH on the terminal Gal residue of a Gal epitope(see, e.g., Andreana et al., Glycoconjugate J., 20: 107-118 (2004)).Exemplary protecting groups include amine (e.g., aminopyridine) andoxime subsituents (e.g., O-Me-oxime, O-Et-oxime, O-tBu-oxime, O-Bn-oximeand O-allyl-oxime). Alternatively, the polar C-6′ OH group can bereplaced with a nonpolar hydrogen to form a 6-deoxy-α-Gal derivative(see Janczuk et al., Carbohydrate Research, 337: 1247-1259 (2002),incorporated by reference herein). In certain exemplary embodiments, theGal epitope can be modified (e.g., at the C-1 OH) with an amino modifier(e.g., alkyl-NH2 substithent) to facilitate linkage to a targetingmoiety. Binding of anti-Gal antibodies to the modified gal antigen canthen be evaluated using art-recognized methodologies (e.g., ELISA).

(d) Multivalent Adaptor Molecules

It is contemplated that a plurality of peptides or peptide moieties ofthe sort disclosed herein could be connected to create a compositeadaptor molecule with increased avidity or valency. Likewise, a peptideor peptide moiety of an adaptor molecule may be attached to any numberof other polypeptides, such as fluorescent polypeptides, targetingpolypeptides and polypeptides having a distinct therapeutic effect.

II. Methods for Identifying High Affinity Adaptor Molecules

In certain aspects, the invention provides methods for identifying anadaptor molecule with high binding affinity or selectivity. The methodsof the invention comprise (i) at least one selection step to identify ahigh affinity targeting moiety (e.g., a targeting peptide moiety and/orligand peptide moiety), and (ii) and a linking step wherein thetargeting and ligand moieties of the adaptor molecule are linked to formthe adaptor molecule.

In certain embodiments, the methods of the invention employ ribosome ormRNA display as a selection step to identify one or more targetingmoieties of an adaptor molecule. A general overview of ribosome and mRNAdisplay methods is provided by Lipovsek and Pluckthun (J. ImmunologicalMethods, 290: 51-67 (2004)), hereby incorporated by reference in itsentirety. In preferred embodiments, the targeting moiety (e.g., apeptide targeting moiety) of the adapator molecule is identified usingmRNA display. An exemplary mRNA display methodology is depicted in FIG.2. Briefly, a starting library is obtained by, e.g., direct DNAsynthesis or through in-vitro or in-vivo mutagenesis. The doublestranded DNA library is then transcribed in-vitro (e.g., using T7polymerase) and attached to a puromycin-like linker. In vitrotranslation is carried out wherein the puromycin-like linker reacts withthe nascent translation product. The result, after purification, is ahighly diverse (˜10¹³) library of peptide-RNA fusion molecules. Reversetranscription generates a cDNA/RNA hybrid, covalently linked to thetranscribed peptide. This complex is then selected for by using thetarget molecule (in the case of a targeting peptide moiety) or anantibody (in the case of a ligand peptide moiety). Peptides that bindthe target or antibody molecule (e.g., under stringent wash conditions)will be selected, and the cDNA is easily eluted to identify the selectedpeptides. The selection can be performed multiple times to identify highaffinity binders. It should be noted that the selection methodology maybe carried out under conditions such that intramolecular disulfide bondsare present in the peptides during selections. In other embodiments, theformation of disulfide bonds may be prevented, if desired.

In additional or alternative embodiments, the methods of the inventionemploy phage display and/or yeast display techniques as a selection stepto identify one or more targeting (e.g., peptide) moieties of an adaptormolecule. Non-limiting examples of such library screening methods aredescribed, for example, in U.S. Pat. Nos. 7,195,880; 6,951,725;7,078,197; 7,022,479; 5,922,545; 5,830,721; 5,605,793, 5,830,650;6,194,550; 6,699,658, each of which is herein incorporated by referencein its entirety.

In other embodiments, the methods of the invention employ libraries ofsynthetic peptides. Such synthetic peptides can be chemicallysynthesized or enzymatically produced (e.g., by in vitro translationfrom RNA or by enzymatic digestion of preexisting proteins). In someembodiments, a library of synthetic peptides is arrayed on a solidsubstrate (e.g., a glass slide).

In certain embodiments, the methods of the invention employ mRNAlibraries (e.g, mRNA display libraries) that encode randomized peptidescorresponding to a portion of a larger polypeptide that is known tointeract with a particular target or antibody molecule. Exemplary mRNAdisplay libraries are depicted in FIG. 3. For example, a peptide librarymay comprise a population of linear peptide molecules where the aminoacid sequence of the peptides are randomized at one or more amino acidpositions (preferably at least 10 or more amino acid positions) withinthe molecule. This randomized portion of the peptide sequence may beflanked by one or more constant regions from the parent polypeptide.

In certain embodiments, the methods of the invention include providingan mRNA display library that encodes a randomized population ofcandidate targeting moieties (e.g., or peptides or polypeptides). By wayof example the targeting peptides may be randomized peptides derivedfrom a soluble ligand, e.g., VEGF. High affinity targeting peptides areselected by screening peptide-RNA-cDNA fusions from the library.Multiple selection cycles are preferably performed to enrich thepopulation of molecules for those that bind to the target molecule(e.g., VEGF receptor). By decreasing the concentration of targetmolecule in each selection step, the peptides which bind to the targetmolecule with highest affinity can be further enriched in thepopulation. Additional selection procedures that can be employed in eachselection step include: (1) contra-selection to eliminate non-specificpeptides; (2) competitive elution to identify site-specific peptides;(3) and selection under specific solution conditions (e.g., highlystringent wash conditions) to identify stable peptides.

In certain embodiments, the members of a library are modified prior toselection to include a coupling moiety which is capable of reacting witha linker (e.g., a bifunctional linker) to form a linking moiety. Inother embodiments, the members of the library are modified with acoupling moiety after the selection step to facilitate linkage to thelinking moiety. Examplary coupling moieties include terminal amino acids(e.g., C- or N-terminal cysteines or cysteine analogs) or amino acidside chains (e.g., cysteine or cysteine analog side chains) which arecapable of reacting with a bifunctional linker of maleimidefunctionality.

Once a high-affinity targeting moiety has been identified, the peptidecan then be linked via a linking moiety to a pre-selected ligand moiety,thereby creating an adaptor molecule. In certain embodiments, the ligandmoiety has been pre-selected using mRNA display methods. Alternatively,the targeting moiety can be inserted as a constant region within asecond mRNA display library that encodes a randomized population ofcandidate ligands. This second mRNA display library can then besubjected to further selection steps wherein the library members arescreened against an antibody to identify an adaptor molecule. Thus, thecandidate ligands preferably correspond to a portion (e.g., an epitope)of an antibody ligand. In one embodiment, an antibody ligand portion canbe an epitope of an antigen to which an antibody binds. In anotherembodiment, the antibody ligand portion can be an idiotope of a firstantibody to which a second, anti-idiotypic, antibody binds. In yetanother embodiment, the antibody ligand portion can be an Fc bindingportion of an Fc binding protein (e.g., an Fc receptor).

Having selected an adaptor molecule that binds with high affinity and/orselectivity to both a target molecule and an antibody molecule, theadaptor molecules can be evaluated for ability to redirect antibodyspecificity to the target molecule. For example, wherein the targetmolecule is cell surface molecule, the ability of the adaptor moleculeto induce an effector function (e.g., antibody-dependent cellularcytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) and cellkilling can be evaluated using art-recognized techniques.

In yet other embodiments, the members of a library are linked to aligand moiety prior to the selection step. For example, each member ofthe library to be screened can be derivatized with a Gal antigen andthen subjected to a screening step to identify a high affinity adaptormolecule. Said Gal antigens may be linked to terminal amino acids oramino acid side chains of each peptide.

In yet other embodiments, an iterative selection process may be employedwherein a targeting moiety is selected in a first selection step (orfirst series of selection steps) and the sequence of the targetingmoiety is incorporated into the constant region of an mRNA-peptidefusion to facilitate the selection of a ligand moiety in a secondselection step (or second series of selection steps). For example, thefirst and second selection steps (or series of selection steps) can bealternated in consecutive rounds of selection to identify high affinityadaptor molecules.

III. Methods for Synthesizing a High Affinity Adaptor Molecule.

Once the components of a high affinity adaptor molecule have beenidentified using the methods described herein, they may be producedusing standard methods known in the art. For example, peptides may beproduced by recombinant DNA methods, inserting a nucleic acid sequence(e.g., a cDNA) encoding the polypeptide into a recombinant expressionvector and expressing the DNA sequence under conditions promotingexpression. General techniques for nucleic acid manipulation aredescribed for example in Sambrook et al., Molecular Cloning: ALaboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology(Green Publishing and Wiley-Interscience: New York, 1987) and periodicupdates, herein incorporated by reference. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts can be found in Cloning Vectors: A Laboratory Manual,(Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference. Other recombinant DNA methods are describedin U.S. Pat. Nos. 4,356,270 4,399,216, 4,506,013, 4,503,142, 4,952,682,5,618,676, 5,854,018, 5,856,123, 5,919,651, and 6,455,275, which are allincorporated herein by reference.

Adaptor molecules and their components may also be made by chemicalsynthesis, using techniques that are well-known in the art. For example,D-peptides can be synthesized using stepwise addition of D-amino acidsin a solid-phase synthesis method involving the use of appropriateprotective groups. Solid phase peptide synthesis techniques commonlyused for L-peptides are described by Meinhofer, Hormonal Proteins andPeptides, vol. 2, (New York 1983); Kent, et al., Ann. Rev. Biochem.,57:957 (1988); Bodanszky et al., Peptide Synthesis, (2d ed. 1976);Atherton et al. (1989) Oxford, England: IRL Press. ISBN 0199630674;Stewart et al. (1984). 2nd edition, Rockford: Pierce Chemical Company,91. ISBN 0935940030; and Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154 all of these references are incorporated herein by reference.D-amino acids for use in the solid-phase synthesis of D-peptides can beobtained from a number of commercial sources. D-peptides and peptidesthat contain mixed L- and D-amino acids are known in the art. Also,peptides containing exclusively D-amino acids (D- peptides) have beensynthesized. See Zawadzke et al., J. Am. Chem. Soc., 114:4002-4003(1992); Milton et al., Science 256: 1445-1448 (1992). Additional methodsto make D-peptides have been described in the art and can be found atleast in WIPO Publication No. WO/1997/013522, and U.S. Application No.60/005,508, which are both incorporated herein by reference.

The peptide of the present invention can be purified byisolation/purification methods for proteins generally known'in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, the peptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis. Thepurified polypeptide is preferably at least 85% or 90% pure, morepreferably at least 93% or 95% pure, and most preferably at least 97%,98%, or 99% pure. Regardless of the exact numerical value of the purity,the peptide is sufficiently pure for use as a pharmaceutical product.

Exemplification

The present disclosure is further illustrated by the figures and thefollowing examples, which should not be construed as further limiting.The contents of all figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference in their entireties.

EXAMPLE 1 Selection of High Affinity Anti-VEGF Peptides as TargetingMoiety and Conjugation to an αGal Ligand Moiety

Using the mRNA display method depicted in FIG. 2, a library ofrandomized peptide sequence were screened for high affinity binding toVEGF. Four high affinity anti-VEGF peptide sequences (SEQ ID NOs 1-4)were selected. Each peptide was PEGylated at the C-terminus withPEG₂-NH2 or PEG₂-Cys-NH2 moiety (see FIG. 4) to facilitate chemicalcoupling to Gal antigen (Bdi-(CH2)-NH2, disacchride).

To facilitate chemical conjugation of each peptide to the Galdisaccharide, a bifunctional linker with maleimide and sulfo-NHSfunctionalities (Sulfo-SMCC, PIERCE) was employed. The linker wasreacted with the amino group present in the disaccharide and a maleimidefunctionality in a sulfhydroxyl group of the C-terminal cysteineresidues of the peptide. To obtain the desired compound and avoid anyfurther reaction and formation of by-products, the reaction wassubjected to RP-HPLC purification right after incubation. Ratios foramounts of reactants were optimized to avoid formation of by-products.In an exemplary synthesis, 6 mg Disaccharide compund B_(di)—(CH₂)—NH₂(˜15 μmol) was dissolved in 100μl 0.1 M Hepes buffer containg 20% MeCN,pH 6.0; 1.3 mg Sulfo-SMCC Linker (˜3 μmol) was dissolved in 100 μl 0.1 MHepes buffer containing 20% MeCN, pH 6.0; and both solutions were mixedand incubated at room temperature for 30 min under continuous rotationof the reaction tube. 1 mg peptide 07-090 (07-090; lyophilisate, TFAadduct, M.W. 3474 g/mol; ˜320 nmol) was dissolved in 1 ml H₂O/MeCN80:20%. Immediately after preparation the clear peptide solution wasadded to the Linker-Disaccharide solution and incubated at roomtemperature for additional 90 min under continuous rotation of thereaction tube. The reaction mixture was put on ice and an aliquot wasanalyzed on a RP-HPLC-C18 column. The desired compound(B_(di)—(CH₂)—NH-Linker-S-07-090-peptide) was purified by running a HPLCgradient from 0 to 50%; buffer A: H₂O/5% MeCN, 0.1% TFA, buffer B:H₂O/5% MeCN, 0.1% TFA. HPLC fractions were analyzed after dilution with65% Methanol, 0.5% formic acid by ESI-TOF-MS analysis and productfractions identified. Relevant product peak fractions were frozen at−80° C. and subsequently lyophilized to complete dryness to harbor thedesired compound.

EXAMPLE 2 Anti-VEGF-Specific Adaptor Molecules Can Redirect NaturalAnti-αGal Antibodies to VEGF

An assay was designed to test the ability of αGal-linked anti-VEGFpeptides to redirect natural antibodies, specific for αGal, to bind toVEGF. The assay was designed with recombinant VEGF on the solid phase. Adilution series of αGal-linked anti-VEGF peptides was then incubatedwith the rVEGF followed by an incubation with sera from mice containinghigh levels of anti-αGal. The amount of bound anti-αGal was thenindicated by an enzyme conjugated anti-mouse antibody. The presence ofenzyme was indicated by the addition of a colorometric substrate and theincrease in color was measured by determining the optical density at 490nm. The data, set forth in FIG. 8, clearly shows that the αGal-linkedanti-VEGF peptides could redirect natural anti-αGal antibodies to VEGFsince a decrease in the amount of peptide or antisera resulted in adecrease in the optical density.

1. A method for identifying a high affinity adaptor molecule capable ofredirecting antibody specificity, the method comprising: (a) providing arandomized library encoding a population of candidate targetingpeptides; (b) selecting a targeting peptide from the display librarywhich binds with high affinity and/or selectivity to a target molecule;(c) linking the targeting peptide to a ligand moiety via a linkingmoiety to form a candidate adaptor molecule; and (d) evaluating theability of the candidate adaptor molecule to redirect the specificity ofthe circulating antibody to the target molecule; thereby identifying theadaptor molecule.
 2. The method of claim 1, wherein steps (a)-(d) areperformed consecutively.
 3. The method of claim 1, wherein linking step(c) is performed prior to step (b).
 4. The method of claim 1, whereinthe library is an mRNA display, ribosome display, yeast display, phagedisplay or synthetic peptide library.
 5. The method of claim 1, whereinthe targeting peptide binds to the target molecule with a bindingaffinity of 1 nM or lower.
 6. The method of claim 1, wherein the ligandmoiety comprises a glycan moiety.
 7. The method of claim 1, wherein theligand moiety is a blood group antigen.
 8. The method of claim 1,wherein the ligand moiety is a gal antigen or epitope thereof.
 9. Themethod of claim 8, wherein the ligand moiety consists of one or moregal-α-1-3-gal disaccharide units.
 10. The method of claim 8, wherein theligand moiety is a modified gal antigen having modifications whichreduce competitive binding by interfering molecules.
 11. The method ofclaim 8, wherein the ligand moiety is a modified gal antigen havingmodifications which reduce enzymatic or chemical degradation.
 12. Themethod of claim 10, wherein the modified gal antigen comprises aprotecting group at a C6′ position of a terminal galactose residue. 13.The method of claim 1, wherein the ligand moiety is a peptidomimetic ofa gal antigen.
 14. The method of claim 1, wherein the ligand moiety is apeptide ligand moiety.
 15. The method of claim 14, wherein the peptideligand moiety comprises an epitope that is selectively bound by anantigen binding site of the circulating antibody.
 16. The method ofclaim 15, wherein the peptide ligand moiety comprises an idiotope of anantibody, wherein the idiotope is selectively bound by a circulatinganti-idiotypic antibody.
 17. The method of claim 16, wherein the peptideligand moiety comprises a binding site portion of an Fc binding protein.18. The method of claim 1, wherein the peptide ligand moiety is selectedby (i) providing a randomized mRNA display library encoding a populationof candidate peptide ligand moieties; and (ii) selecting a peptideligand moiety from the display library of step (i) which binds with highaffinity and/or selectivity to a circulating antibody.
 19. The method ofclaim 18, wherein the candidate peptide ligand moieties are fused totargeting peptides prior to selection step (ii).
 20. The method of claim18, wherein the candidate peptide ligand moieties are fused to targetingpeptides following selection step (ii).
 21. The method of claim 1,wherein the target molecule is a soluble disease-associated molecule.22. The method of claim 21, wherein the redirected antibody specificityis evaluated by measuring opsonization or neutralization of the solublemolecule.
 23. The method of claim 1, wherein the ligand moiety is linkedto the targeting moiety with a bifunctional linker moiety.
 24. Themethod of claim 23, wherein the bifunctional linker moiety links thetargeting moiety and the ligand moiety via an amino group in thetargeting moiety and a thiol moiety in the ligand moiety.
 25. The methodof claim 1, wherein the target molecule is present on the surface of aninfected or neoplastic cell.
 26. The method of claim 25, wherein theredirected antibody specificity is evaluated by measuring ADCC orCDC-dependent killing of the cell.
 27. A high affinity adaptor moleculeidentified according to the method of claim 1, the adaptor moleculecomprising (i) a targeting moiety which binds with high affinity orselectivity to a target molecule, (ii) a ligand moiety whichspecifically binds to a circulating antibody; and (iii) a linker moietylinking the targeting moiety to the ligand moiety, wherein the adaptormolecule facilitates a functional interaction between the antibody andthe target molecule.
 28. The high affinity adaptor molecule of claim 27,wherein the targeting moiety is a peptide targeting moiety.
 29. Theadaptor molecule of claim 27, wherein the targeting moiety binds withhigh affinity or selectivity to VEGF ligand.
 30. The adaptor molecule ofclaim 27, wherein the targeting moiety comprises one or more sequencesselected from the group consisting of SEQ ID NOs 1, 2, 3 and
 4. 31. Theadaptor molecule of claim 27, wherein the targeting moiety is PEGylated.32. The adaptor molecule of claim 27, wherein the ligand moietycomprises a Gal antigen which specifically binds to a circulatinganti-Gal antibody.
 33. A high affinity adaptor molecule selected fromthe group consisting of:(a) H-Gly-D-Val-D-Gln-D-Glu-D-Asp-D-Val-D-Ser-D-Ser-D-Thr-D-Leu-Gly-D-Ser-D-Trp-D-Val-D-Leu-D-Leu-D-Pro-D-Phe-D-His-D-Arg-Gly-D-Thr-D-Arg-D-Leu-D-Ser-D-Val-D-Trp-D-Val-D-Thr-PEG2-Cys-X-Y;(b) H-Gly-Gly-D-Phe-D-Glu-Gly-D-Leu-D-Ser-D-Gln-D-Ala-D-Arg-D-Lys-D-Asp-D-Gln-D-Leu-D-Trp-D-Leu-D-Phe-D-Leu-D-Met-D-Gln-D-His-D-Ile-D-Arg-D-Ser-D-Tyr-D-Arg-D-Thr-D-Ile-D-Thr-PEG2-Cys-X-Y;(c) H-Gly-D-Val-Gly-Gly-D-Ser-D-Arg-D-Leu-D-Glu-D-Ala-D-Tyr-D-Lys-D-Lys-D-Asp-D-His-D-Arg-D-Val-D-Phe-D-Gln-D-Met-D-Ala-D-Trp-D-Leu-D-Gln-D-Tyr-D-Tyr-D-Trp-D-Ser-D-Thr-D-Thr-PEG2-Cys-X-Y; and(d) H-Gly-D-Ser-Gly-D-Ser-Gly-D-Asn-D-Ala-D-Leu-D-His-D-Trp-D-Val-D-Cys-D-Ala-D-Ser-D-Asn-D-Ile-D-Cys-D-Trp-D-Arg-D-Thr-D-Pro-D-Trp-D-Ala-Gly-D-Gln-D-Leu-D-Trp-Gly-D-Leu-D-Val-D-Arg-D-Leu-D-Thr- PEG2-Cys-X-Y;

wherein, X is bifunctional chemical linker with maleimide functionality;and Y is an amino modified Gal-1-3-Gal disaccharide.